Homocitrate Synthase from Penicillium Chrysogenum. Localization, Purification of the Cytosolic Isoenzyme, and Sensitivity to Lysine

Overview

Journal Biochem J

Specialty Biochemistry

Date 1990 Jul 1

PMID 2115771

Citations 11

Authors

W M Jaklitsch

C P Kubicek

Affiliations

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Abstract

Subcellular fractionation of cell-free extracts obtained by nitrogen cavitation showed that Penicillium chrysogenum Q176 contains a cytosolic as well as a mitochondrial homocitrate synthase activity. The cytosolic isoenzyme was purified about 500-fold, and its kinetic and molecular properties were investigated. Native homocitrate synthase shows a molecular mass of 155 +/- 10 kDa as determined by gel filtration and a pH of 4.9 +/- 0.1 as determined by chromatofocusing. The kinetic behaviour towards 2-oxoglutarate is hyperbolic, with Km = 2.2 mM; with respect to acetyl-CoA the enzyme shows sigmoidal saturation kinetics, with [S]0.5 = 41 microM and h = 2.6. The enzyme was inhibited strongly by L-lysine (Ki = 8 +/- 2 microM; 50% inhibition by 53 microM at 6 mM-2-oxoglutarate), competitively with 2-oxoglutarate, in protamine sulphate-treated and desalted cell-free extracts and in partially purified preparations. The extent of this inhibition was strongly pH-dependent. Both isoenzymes are equally susceptible to inhibition by lysine. The same inhibition pattern is shown by the enzyme from strain D6/1014A, which is a better producer of penicillin than strain Q176.

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References

Masurekar P, DEMAIN A . Insensitivity of homocitrate synthase in extracts of Penicillium chyrosogenum to feedback inhibition by lysine. Appl Microbiol. 1974; 28(2):265-70. PMC: 186699. DOI: 10.1128/am.28.2.265-270.1974. View

Tucci A, CECI L . Homocitrate synthase from yeast. Arch Biochem Biophys. 1972; 153(2):742-50. DOI: 10.1016/0003-9861(72)90393-1. View

Tracy J, Kohlhaw G . Reversible, coenzyme-A-mediated inactivation of biosynthetic condensing enzymes in yeast: a possible regulatory mechanism. Proc Natl Acad Sci U S A. 1975; 72(5):1802-6. PMC: 432634. DOI: 10.1073/pnas.72.5.1802. View

Gaillardin C, Poirier L, Heslot H . A kinetic study of homocitrate synthetase activity in the yeast Saccharomycopsis lipolytica. Biochim Biophys Acta. 1976; 422(2):390-406. DOI: 10.1016/0005-2744(76)90150-9. View

Bradford M . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72:248-54. DOI: 10.1016/0003-2697(76)90527-3. View

Friedrich C, DEMAIN A . Uptake and metabolism of alpha-aminoadipic acid by Penicillium chrysogenum Wis 54-1255. Arch Microbiol. 1978; 119(1):43-7. DOI: 10.1007/BF00407926. View

Luengo J, Revilla G, VILLANUEVA J, Martin J . Lysine regulation of penicillin biosynthesis in low-producing and industrial strains of Penicillium chrysogenum. J Gen Microbiol. 1979; 115(1):207-11. DOI: 10.1099/00221287-115-1-207. View

Luengo J, Revilla G, Lopez M, VILLANUEVA J, Martin J . Inhibition and repression of homocitrate synthase by lysine in Penicillium chrysogenum. J Bacteriol. 1980; 144(3):869-76. PMC: 294747. DOI: 10.1128/jb.144.3.869-876.1980. View

Merril C, Goldman D, Sedman S, EBERT M . Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science. 1981; 211(4489):1437-8. DOI: 10.1126/science.6162199. View

10.

Osmani S, Scrutton M . The sub-cellular localisation of pyruvate carboxylase and of some other enzymes in Aspergillus nidulans. Eur J Biochem. 1983; 133(3):551-60. DOI: 10.1111/j.1432-1033.1983.tb07499.x. View

11.

Osmani S, Scrutton M . The sub-cellular localisation and regulatory properties of pyruvate carboxylase from Rhizopus arrhizus. Eur J Biochem. 1985; 147(1):119-28. DOI: 10.1111/j.1432-1033.1985.tb08727.x. View

12.

Bhattacharjee J . alpha-Aminoadipate pathway for the biosynthesis of lysine in lower eukaryotes. Crit Rev Microbiol. 1985; 12(2):131-51. DOI: 10.3109/10408418509104427. View

13.

Jaklitsch W, Hampel W, Rohr M, Kubicek C, Gamerith G . alpha-Aminoadipate pool concentration and penicillin biosynthesis in strains of Penicillium chrysogenum. Can J Microbiol. 1986; 32(6):473-80. DOI: 10.1139/m86-087. View

14.

Affenzeller K, Jaklitsch W, Honlinger C, Kubicek C . Lysine biosynthesis in Penicillium chrysogenum is regulated by feedback inhibition of alpha-aminoadipate reductase. FEMS Microbiol Lett. 1989; 49(2-3):293-7. DOI: 10.1016/0378-1097(89)90056-6. View

15.

Honlinger C, Kubicek C . Regulation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine and isopenicillin N biosynthesis in Penicillium chrysogenum by the alpha-aminoadipate pool size. FEMS Microbiol Lett. 1989; 53(1-2):71-5. DOI: 10.1016/0378-1097(89)90368-6. View

16.

Hogg R, BROQUIST H . Homocitrate formation in Neurospora crassa. Relation to lysine biosynthesis. J Biol Chem. 1968; 243(8):1839-45. View

17.

Betterton H, Fjellstedt T, Matsuda M, OGUR M, Tate R . Localization of the homocitrate pathway. Biochim Biophys Acta. 1968; 170(2):459-61. DOI: 10.1016/0304-4165(68)90036-6. View

18.

Laemmli U . Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227(5259):680-5. DOI: 10.1038/227680a0. View

19.

DEMAIN A, Masurekar P . Lysine inhibition of in vivo homocitrate synthesis in Penicillium chrysogenum. J Gen Microbiol. 1974; 82(1):143-51. DOI: 10.1099/00221287-82-1-143. View